We have recently established that the cyclic redox phenothiazinium chromophore methylene blue (MB), already approved for the treatment of methemoglobinemia, counteracts the toxicity of cyanide (CN). MB produces a dramatic increase in survival rate following an otherwise lethal CN exposure and can restore the cardiac function during CN-induced acute cardiac failure. We also found that the mechanism of action of MB is unique among the currently proposed CN antidotes, relying on its redox properties. MB can restore the Krebs cycle, through its oxidizing effects on NADH, as the mitochondrial electron chain activity is inhibited by CN. MB can also rescue the mitochondrial gradient potential of CN poisoned cells through a mechanism that is not yet fully understood. These effects translate into an immediate recovery of intoxicated cardiomyocytes and into the correction of CN-induced circulatory shock with a drastic decrease in mortality and neurological sequelae. Such effects may also explain why MB is effective against other mitochondrial poisons such as H2S. MB is metabolized in vivo into the mono-demethylated Azure B (AzB), a molecule that we have also found to be present in commercial solutions of MB. AzB, in contrast to the other demethylated metabolites of MB, appears to possess redox properties similar to those of MB. Preliminary data accumulated over the last 2 years, along with results from the literature, support the view that AzB is as well tolerated as MB after systemic administration, while displaying a higher efficacy in certain conditions associated with mitochondrial dysfunction. For instance, AzB restores blood pressure and VO2 more readily and at higher level than MB after sulfide intoxication. The superiority of AzB could be, in part, accounted for by the electrical neutrality of its oxidized form allowing for a better diffusion than MB through cellular membranes. These findings prompted us to develop this proposal. Our objective is to establish whether our preliminary observations suggesting a superiority of AzB over MB are clinically meaningful during lethal or sub-lethal CN intoxications. We will compare the antidotal properties of AzB and MB, administered after CN exposure, in two complementary animal models of CN intoxication in the rat. A first model will allow the study of immediate survival and of the short and long-term outcomes after a lethal exposure to CN in awake rats. The second model, developed in sedated rats, will produce a sub-lethal CN intoxication, wherein the effects of AzB vs MB on cardiac contractility and breathing pattern will be investigated. The study will also look at the recovery of VCO2, a marker of the Krebs cycle, as well as the pyruvate/lactate and acetoacetate/beta-hydroxybutyrate ratios, surrogates for NADH/NAD ratio in the cytoplasm and the mitochondria respectively. If the superiority of AzB over MB is confirmed, our final objective is to develop a robust plan for additional safety and efficacy studies aimed at offering AzB, alone or in combination with other CN antidotes, as a pre- hospital treatment of severe intoxications by CN and possibly other mitochondrial poisons. !